1. Field of the Invention
The present invention is directed to DNA transformation constructs encoding mobile elements and their use for transforming eukaryotic cells. In particular transposons are used as a mechanism for inserting DNA sequences into the cell""s genome after introduction of the transformation construct into the cell.
2. Description of the Related Art
Certain natural DNA sequences in eukaryotic and prokaryotic cells have the ability to move from one genomic locus to a second locus. These genetic elements are referred to generally as transposable elements or transposons. Advantageously, these transposable elements can be used as tools for genetically manipulating cells. In particular, transposable elements isolated from eukaryotes are anticipated as having the greatest potential for use in producing transgenic organisms.
Transposable elements can be divided into two classes. Class I are the retro-transposons that replicate through an RNA intermediate and utilize reverse transcriptase to produce a DNA molecule that is inserted into the host cell""s genome. The Class II transposons include all other mobile elements and include P, hobo, mariner, Tcl, and Ac elements (Berg and Howe, Mobil DNA, American Society for Microbiology, Washington, D.C. 1989). Members of this transposon class have short inverted repeats at their termini and generate direct duplications of a host target sequence upon insertion. Many of these elements are currently being developed as general transformation vectors in insects and plants (Rubin and Spradling, Science, Volume 218, 348-353 1982; Lidholm, Lohe and Hartl, Genetics, Volume 134, 859-868 1993; O""Brochta and Handler, Prospects and possibilities for gene transfer techniques in insects, 451-488; in Molecular Approaches to Fundamental and Applied Entomology, ed. Oakeshott et al, Springer-Verlag, New York, 1993).
The P element has been used effectively for Drosophila transformation but has limited use as a general transformation vector because it is not active in species other than Drosophila (O""Brochta and Handler, 1993 supra; Rubin and Spradling, 1982 supra). The mariner element is phylogenetically dispersed (Robertson, H. Insect Physiol., Volume 41, 99-105, 1995), and therefore apparently has the capability of movement in a number of diverse species. In addition, the hobo element has demonstrated mobility in diverse genetic backgrounds and is a promising candidate for development as a genetic engineering tool (Atkinson, Warren and O""Brochta, PNAS USA, Volume 90, 9693-9697 1993; O""Brochta and Handler, 1993 supra; O""Brochta et al., Mol. Gen. Genet., Volume 244, 9-14, 1994).
PiggyBac (previously described as IFP2) and tagalong elements are unique Lepidopteran transposons structurally related to the Class II DNA transposable elements (Finnegan, Curr. Opin, Cell Bio., Volume 2, 471-477 1990). These transposons were isolated from the cabbage looper moth, Trichoplusia ni Hubner (Lepidoptera: Noctuidae). The piggyBac element was first identified as an insertion within Galleria mellonella or Autographa californica nuclear polyhedrosis virus genomes following passage of the viruses in the Trichoplusia ni insect cell line, TN-368. (Fraser et al., Virology, Volume 145, 356-361, 1985; Fraser et al., J. Virology, Volume 47, 287-300, 1983).
The piggyBac and tagalong elements are unusual among Class II transposons in that those elements always target and duplicate the tetranucleotide, TTAA, upon insertion in Baculovirus-infected cells (Cary et al., Virology, Volume 172, 156-169, 1989). The specificity for TTAA target sites is exhibited by other Lepidopteran transposon-like insertions as well (Beames and Summers, Virology, Volume 162, 206-220 1988; Beames and Summers, Virology, Volume 174, 354-363 1990; Carstens, Virology, Volume 161, 8-17, 1987; Oellig et al., J. Virology, Volume 61, 3048-3057, 1987; Schetter, Oellig and Doerfler, J. Virology, Volume 64, 1844-1850, 1990). Thus the piggyBac and tagalong elements are part of a subclass of the Class II transposons.
In addition to TTAA target specificity, all Lepidopteran transposons having the TTAA target specificity terminate in at least two C residues at the 5xe2x80x2 ends of their inverted repeats. Given their similarity in insertion site selection and duplication, all of these TTAA specific elements are likely to excise in a similar manner.
Furthermore piggyBac and tagalong elements excise precisely upon transposition in vivo, leaving behind the single TTAA target sequence upon excision. The excision events of piggyBac and tagalong are dissimilar to the transposase-associated excision events of the hAT family of transposons. This family includes hobo, hermes, Ac and Tam3. (Calvi et al., Cell, Volume 66, 465-471, 1991). Elements in the hAT family vary in the length and nucleotide sequence of their inverted terminal repeats (Calvi et al., 1991; supra), but have a conserved A2G5 motif within these repeats, and generate 8 bp target site duplications (Warren et al., Genet. Research, Volume 64, 87-97, 1994). These elements excise imprecisely in the presence of an element-encoded transposase and leave behind characteristic footprints that have proven useful in distinguishing transposase-associated excision events (Atkinson et al., 1993 supra; Warren et al., 1994 supra).
Most of the transposase-associated excisions of P-elements are imprecise events, leaving behind part or all of the 31 bp terminal inverted repeat and adding xe2x80x98fillerxe2x80x99 sequences at the excision breakpoints (O""Brochta et al, Mol. Gen. Genet., Volume 225, 387-394, 1991: Takasu-Ishikawa et al., Mol. Gen. Genet., Volume 232, 17-23, 1992). In the case of the hobo element of Drosophila melanogaster, excision from plasmids in microinjected fertile eggs most often involves the complete removal of hobo and some flanking nucleotides with the addition of filler sequences related to flanking host DNA at the excision breakpoints (Atkinson, Warren and O""Brochta, 1993 supra; Handler and Gomez, Mol. Gen. Genet., Volume 247, 399-408 1995; O""Brochta and Handler, 1993 supra).
In contrast with these other Class II elements, precise excision of piggyBac and tagalong is the rule rather than the exception. Precise excision of genetically tagged piggyBac elements was first demonstrated in Baculovirus genomes of infected cells (Fraser et al, Virology 211, 397-407 1995). However, the precise excision of the piggyBac element has also been demonstrated in non-virus infected cells indicating the excision of piggyBac is not dependant on Baculovirus protein products. The frequency of precise excision events in transiently transfected IPLB-SF21AE cells is greatly enhanced by the presence of a helper element encoding a full-length transposase. The excision event is believed to be a non-conservative event involving double-strand breaks at or near the transposon termini.
The present invention, discussed below, provides recombinant DNA vectors derived from the piggyBac and tagalong transposons which are different from related art vectors. Furthermore, the present invention provides a method to produce transgenic organisms using the recombinant DNA vectors. The transposon genetic transformation system of the present invention provides vectors and broad spectrum methods for the introduction of foreign genes that do not currently exist.
It is therefore an object of the present invention to provide DNA sequences capable of allowing for almost precise excision of a second DNA sequence inserted into a plasmid and insertion of said second DNA sequence into a host cell after transformation of said host cell with a transformation construct containing said first and second DNAs.
Another object of the present invention is to provide transformation constructs including DNA derived from a piggyBac transposon element which allow for the almost precise excision of a second DNA sequence included in the construct and insertion of said second DNA sequence into a host cell after introduction of a transformation construct containing said first and second DNAs into said host cell.
A further object of the present invention is to provide a transformation construct containing transposing elements combined with a DNA sequence capable of being expressed in a transformed host cell.
A still further object of the present invention is to provide a DNA sequence capable of being expressed in a transformed cell flanked by piggyBac or tagalong terminal inverted repeats.
Another object of the present, invention is to provide a method for making a transgenic organism by inserting a transformation construct containing a DNA sequence, capable of being expressed in a transformed cell, flanked by piggyBac or tagalong inverted repeats into a cell; wherein the DNA sequence will excise from the construct and will insert into the host cell at least at the target sequence TTAA in said host cell genome and using the transformed cell to obtain said transgenic organism.
Further objects and advantages of the present invention will become apparent from the following description.